003 I -8655/79/0501-0991 ~ 2 . 0 0 1 0
Photochemistry ond Phorobralogy. Vol. 29. pp. 991 10 IMW). 0 Perg,,rnun Press Lld. 1979. Printcd in Great Brilmn
VISIBLE LIGHT IRRADIATION OF HUMAN AND BOVINE SERUM ALBUMIN-BILIRUBIN COMPLEX* FIRMINO F. RUBALTELLI and G~ULIO JORI Department of Pediatrics and C.N.R. Center for the Physiology and Biochemistry of Hemocyanins and Other Metallo-Proteins, Institute of Animal Biology, University of Padova, Padova, Italy (Received 12 October 1978; accepted 14 December 1978)
Abstract-The UV absorption and the fluorescence emission spectra of both bovine (BSA) and human (HSA) serum albumin underwent noticeable changes upon irradiation of their 1: 1 complexes with bilirubin; both these phenomena are suggestive of the photosensitized modification of aromatic amino acid residues. Amino acid analysis showed that after relatively short irradiation times of both albumins, only histidyl and tryptophyl residues appeared to be affected to a significant extent. After 60min of irradiation, some decrease in the tyrosine content was also observed, especially for HSA. Conformational studies, obtained by exposing unirradiated and irradiated BSA and HSA to denaturing agents, showed that the three-dimensional organization of the 15 min irradiated samples was slightly differentfrom that of the native proteins. On the other hand, after 15 min of irradiation, the association constant of the bilirubin-albumin complexes decreased from 2.07 to 0.54 x IO'M-' for HSA and from 2.16 to 0.87 x LO'M-' for BSA. These data indicate that the histidyl residues are relatively unimportant for maintaining the native tertiary structure of BSA and HSA, but they are critical for determining the binding capacity of the albumins. Our data also imply that the tertiary structure of the BSA molecule is more labile than that of HSA. INTRODUCTION
Phototherapy is frequently used in the treatment of unconjugated hyperbilirubinemia of premature newborns (Rubaltelli, 1973; Sisson, 1976). Present evidence indicates that photodynamic damage can be ruled out as a major hazard of in uiuo light treatment, although some metabolic processes may be altered in jaundiced infants treated by phototherapy (Rubaltelli and Largajolli, 1973; Rubaltelli et al., 1974; Bakken, 1977). However, it is still to be shown that minor photodynamic effects are absent. Since bilirubin is predominantly bound to albumin, it is conceivable that amino acid residues of the albumin molecule are, at least in uitro, the main site for photodynamic attack. Photooxidized albumin exhibits a reduced bilirubin-binding capacity (Cashore et al., 1975a; Odell et al., 1970), which might possibly increase the risk of kernicterus. Cashore et al. (1975b) were able t o demonstrate that phototherapy does not influence the bilirubinalbumin complex in uiuo, at least as studied with Sephadex gel filtration. However, the effect of light treatment on albumin in the presence of bilirubin has not been fully clarified. The pioneering work of Odell et al. (1970) has shown that, in the presence of sensitizing dyes, the imidazole ring of histidine is readily destroyed by light treatment in uitro and, in addition, the bilirubin-albumin binding is decreased, possibly *This work has been supported in part by C.N.R. grants No. 77.01550.64 and No. 78.01876.65 under the C.N.R.N.S.F. cooperative research program.
as a cokequence of alterations of the albumin tertiary structure. O n the contrary, Pedersen et al. (1977) have found that human serum albumin does not undergo in uitro photooxidation upon direct irradiation of its complex with bilirubin and have proposed that one of the possible mechanisms of phototherapy is a Z-E photoisomerization of bilirubin when complexed with albumin to produce a water-soluble compound. Most authors generally agree that the albuminbound bilirubin is photooxidized at a slower rate than unbound bilirubin, although the mechanism of such photoprotection is not fully understood (Lightner, 1977). O n the other hand, Ostrow and Branham (1970) and Ostrow (1972) found that at pH values from 7.4 to 9.9 the albumin addition accelerates the destruction of bilirubin by light, whereas the protein stabilizes the pigment against light at pH 11-13. The present studies were designed to investigate the photodegradation of the bilirubin-albumin complex upon direct irradiation with visible light, as well as upon irradiation in the presence of different photosensitizers. The eventual changes of the bilirubinalbumin complex were also studied. MATERIALS AND METHODS
Materials, Bovine (BSA) and human (HSA) serum albumin, crystallized and lyophilized, Fraction V, were pur-
chased from Sigma; their concentrations were estimated spectrophotometrically assuming extinction values at 279 nm of 6.67 and 5.30 for 1% solutions of BSA and HSA, respectively (Clark et al., 1962). Since defatting the proteins with charcoal in acid solution (Chen, 1967) had no appreci591
992
JORI FIRMINO F. RUBALTELLIand GIULIO
able effect on the photooxidation, the commercial preparations were used as such. Bilirubin (Sigma) was shown by thin layer chromatography (McDonagh and Assisi, 1972) to be essentially constituted by the IXa isomer, and its concentration was determined using a molar absorptivity value of 46.500 at 459nm (Chen, 1973). Methylene blue and riboflavin (Merck). acridine orange and hematoporphyrin (Sigma) were used as received. Guanidine hydrochloride (Merck) was recrystallized once from absolute ethanol and fresh solutions of this compound were prepared daily. Bilirubin and bilirubin-protein complexes were prepared by dissolving the dye in a few drops of 0.1 M NaOH and by adding the desired amount of buffer or of a protein solution in 0.5 M KH2PO4-Na2HPO4 buffer at pH 7.4, ionic strength 0.1. The solutions were maintained in the dark and used within a few h. Final solutions to be irradiated were 0.1 mM in both albumin and bilirubin, and absorption spectral studies showed that bilirubin solutions strictly followed Beer's law, suggesting that the dye was present essentially in a monomeric form; under these conditions, bilirubin is essentially all bound at the single high affinity site of HSA and BSA (Jacobsen, 1977).In all cases, the dark controls were recovered unchanged within the periods of time necessary for the irradiation experiments. Irrcidiulion procedure. Light from a stabilized 1250 W high pressure halogen lamp (Osram) was focused by a parabolic reflector on a 1 cm-thick quartz cuvette containing 3m/ of an air-equilibrated aqueous solution of the sample to be irradiated. A freshly prepared acetone filter was placed in the front of the irradiation cuvette to eliminate all wavelengths below 330nm. The irradiance in the 340-470nm region, as measured with a thermopile. was about 3 W/m2/nm. The temperature of the solutions was maintained at 19 f 1°C by means of thermostated water circulating through the cell holder. In kinetic experiments, aliquots were taken at suitable intervals for spectroscopic and other analysical measurements. When the irradiated solutions contained dyes acting as photosensitizers, at a concentration of 0.1 mg/m/, suitable Bakers interference filters with peak transmission at 404 nm (for riboflavin and hematoporphyrin), 490 nm (for acridine orange) and, 640nm (for methylene blue) were used. Spectroscopic measurements. Absorption spectra were monitored at room temperature by a Perkin-Elmer 402 spectrophotometer. using matched quartz cuvettes with an optical path of either 10 or 1 mm. Absorbance measurements at specific wavelengths were obtained with a Zeiss M4/Q 111 single beam spectrophotometer. Fluorescence emission spectra were recorded by a Perkin-Elmer MPF4 spectrophotofluorimeter, equipped with a 150 W Xe light source and R446 F photomultiplier tube. Quartz cuvettes with an optical path of lOmm were used and the light emission was read at 90" to the incident beam. The temperature of the analyzed solutions could be controlled to + O S T by circulating water; in all cases, the samples were kept at the desired temperature for about 10min prior to measurement, in order to ensure the attainment of thermal equilibrium. The optical density of the various samples was always lower than 0.15 at the excitation wavelength, in order to minimize artifacts due to inner filter or selfabsorption effects. Finally, the spectra were corrected for any fluorescence arising from the solvent system used. On the other hand, no corrections were applied for the wavelengthdependence of the light source emission and detector response. Prior to absorption spectral measurements, the dyes and/or bilirubin were removed by a gel filtration of the unirradiated or irradiated solutions through a column (35 x 1.2 cm) of Sephadex (3-25, using the 0.5 M phosphate buffer as the eluant. Control experiments showed that bilirubin was completely removed from unirradiated albumin; on the other hand, after irradiation of the complex, bilirubin and/or
some photoproducts were not removed from albumin even after denaturation of the protein. Further experiments are now in progress in order to clarify the nature of this apparently photoinduced binding. Amino ucid analyses. The dye- and salt-freed albumin solutions were hydrolyzed by heating at 1 1 0 C for 2 h within evacuated sealed vials in the presence of 6 M HCI. The solvent was then removed by lyophilization and the dried samples were chromatographed on a Carlo Erba 3A27 amino acid analyzer following the method of Moore and Stein (1963). The recovery of the various amino acids was estimated using phenylalanine, valine and alanine as internal standards, since these amino acids are known to be stable to acid hydrolysis and are unaffected by photosensitized oxidation processes (Spikes and MacKnipht. 1970). The content of tryptophan was measured on the intact protein by the spectrophotofluorimetric method described elsewhere (Genov and Jori, 1973). To avoid artifacts arising from the effect of protein conformation on the emission properties of the tryptophyl side chains (Longworth, 1971), the protein samples were denatured by diluting 0.5 m/ of the albumin solution with 2.5 m/ of 7 M guanidinium chloride solution in the same buffer. Control experiments showed that there was a linear relationship between protein concentration and intensity of the 295 nmexcited emission. RESULTS
Photodegradation of bilirubin by direct irradiariori Binding of bilirubin with HSA and BSA was accompanied by the well-known bathochromic shift of the absorption maximum of the pigment and by the appearance of hyperchromicity. Irradiation of either free or albumin-bound bilirubin with polychromatic radiation from the halogen lamp caused a gradual decrease of the absorbsnce in the 450 nm region
10
20
30
IRRAOIATION TIME (MIN )
Figure 1. Time-course of the absorbance decrease at 490 nm observed upon irradiation of free bilirubin (.---a), BSA-bound (0-0) and HSA-bound (A-A) bilirubin.
993
Serum albumin-bilirubin complex
-- I . d
40
2.
t
2 30-
* z W
-
W
y
20-
W
w
I
L
680
640
600
I
I
L
560
520
480
WAVELENGTH
Inm)
Figure 2. Fluorescence emission spectra displayed by 0.1 m M bilirubin solutions in 0.5 M phosphate buffer. pH 7.4, which had been irradiated for different periods of time (min). Excitation wavelength: 480 nm. Instrumental Sensitivity: 30; excitation slit: 8 nm: emission slit: 10 nm. and a concomitant enhancement of the absorbance below 350nm, in agreement with the observations of several other authors (see, for a review, Lightner, 1977). The time-course of light induced decrease of bilirubin absorption at 490nm is shown in Fig. 1; this wavelength was chosen since previous studies pointed out that at 490 nm little interference occurs from the bilirubin photodegradation products (Davies and Keohane, 1970; Ostrow, 1972). Essentially identical plots were obtained if the absorbance changes were followed at 460 nm. Clearly, the spectral changes were less pronounced for any given period of irradiation when bilirubin was complexed with HSA or BSA. Further information on bilirubin degradation was obtained by fluorescence emission studies. As shown by other authors (Beaven et a!., 1973; Chen, 1973), binding of bilirubin to albumins results in a remarkI
I
c
A
B
I
20 IRRAOIATION TIME (min 1 10
30 TIME
Figure 3. Time-course of the emission intensity enhancement at 540 nm observed upon irradiation of free bilirubin ( 0 . -0).
able enhancement of the intrinsically low fluorescence of the pigment around 530 nm. We have reconfirmed these data. Moreover, we observed that, upon irradiation, the yield of the 480 nm-excited fluorescence emission increases (see, for example, Fig. 2), although the rate and the extent of the fluorescence enhancement were again different for free and protein-bound bilirubin (Fig. 3); in particular, for HSA-bound bilirubin, no appreciable change of the fluorescence yield was observed after about 10min of irradiation. As pointed out above, the fluorescence quantum yield of free bilirubin is very low; therefore, the emission enhancement should not reflect a dissociation on the bilirubin-albumin complex. Rather, it might be connected with the formation of bilirubin photoisomers
BSA-bound
(0-O),
and
HSA-bound
(A --A)bilirubin under the experimental and instrumental conditions specified for Fig. 2.
(MINI
Figure 4. Time-course of 0.1 mM bilirubin photodegradahematoporphyrin tion sensitized by riboflavin (0-0). (A-A), methylene blue (a-W), and acridine orange (o-o), as deduced from the absorbance decrease at 490 nm. A: free bilirubin, B: BSA-bound bilirubin.
994
FIRMINO
F. RUBALTELLIand GIIJLIO JORI
(McDonagh, 1971; Ostrow, 1972; Kostenbauder and Sanvordeker, 1973; Lightner, 1977). We have also studied the disappearance of free and albumin-bound bilirubin, photosensitized by a variety of dyes, under our experimental conditions (See Fig. 4a and b). Since such irradiation was carried out with narrow light bands and under conditions ensuring total absorption of the incident light by the added dye, one can conclude that BSA also protects bound bilirubin from photosensitized degradation. O n the other hand, it is not possible to compare the kinetic plots obtained with the different photosensitizers, since no correction was applied for the quantum efficiency of the light emission by the halogen lamp as a function of the wavelength.
1001
Bilirubin-sensitized photooxidation of albumins
250
290
270
310
WAVELENGTH (nm)
Figure 5. Ultraviolet absorption spectra of 0.05 mM BSA solutions after 0 (.---a), 15 (0-O), and 60 (A-A) minutes of irradiation of their 1:1 complex with bilirubin. Bilirubin and its photoproducts had been previously removed by gel filtration. (Lightner, 1977; Pedersen et a/., 1977) and/or with the formation of fluorescent bilirubin photodegradation products, which have been actually detected in developed chromatograms of irradiated bilirubin solutions (Ostrow, 1972). Photosensitixd deyrudution of bilirubin
Bilirubin degradation can be photosensitized by the addition of suitable dyes to the irradiated system
The UV absorption spectrum of both BSA and HSA underwent noticeable changes upon prolonged irradiation of their 1:l complexes with bilirubin as shown in Fig. 5: the alterations are suggestive of the photosensitized modification of aromatic amino acid residues (Spikes and Livingston, 1969). The same line of evidence was provided by studies of the intrinsic fluorescence emission of the albumins as a function of the irradiation time: as one can see from Figs. 6a and b, prolonging the light-exposure caused a steady decrease of the emission yield of both BSA and HSA. Since the exciting radiation (295 nm) was selectively absorbed by the tryptophyl chromophores. this phenomenon must be the consequence of a gradual photooxidative modification of the indole side chains and/or of conformational transitions of the albumin molecule leading to perturbations of the microenvironment of the tryptophyl residues. In actual fact, quantitative determination of Trp by spectrophotofluorimetric analysis on the unirradiated and irradiated albumin samples. after denatu-
A
420
380
420
340 WAVELENGTH
380
340
trim)
Figure 6. Fluorescence emission spectra of 0.1 mM HSA (a) and BSA (b) solutions in 0.5 M phosphate buffer, pH 7.4, after irradiation of their 1 : 1 complex with bilirubin. Excitation wavelength: 295 nm. Instrumental sensitivity: I ; excitation and emission slits: 6 nm.
Serum albumin-bilirubin complex Table 1. Tryptophan content of human and bovine serum albumin after different periods of irradiation of their complex with bilirubin* Irradiation time (min) ~~
~
Theoryt
-
0 15 60
Table 2. Amino acid composition of human serum albumin after different periods of irradiation of its complex with bilirubin* ~
~~
Amino acid HSA
BSA
1.o 1 .o 0.8 0.5
2.0 1.9 1.4 0.8
~~
*Determined according to the spectrophotofluorimetric procedure described by Genov and Jori (1973). The values in the table are expressed as mol of amino acid per mole of protein. tTaken from Behrens et 01. (1975) for HSA and from Brown (1975) for BSA. ration by 7 M guanidinium chloride, gave the results summarized in Table 1. O n the basis of the above reported studies, protein samples were isolated after 15 and 60min of irradiation for complete amino acid analysis. The data obtained for the unirradiated samples were compared with available reports of the amino acid sequence and composition of HSA (Behrens et al., 1975) and BSA (Brown, 1975), as revised in the 1976 edition of the Atlas of Protein Sequence and Structure published by the National Biomedical Research Foundation, U.S.A. As one can see from Tables 2 and 3, after relatively short irradiation times of both albumins, only histidyl residues appeared to be affected to a significant extent. After 60 min of irradiation, some decrease in the tyrosine content was also observed, especially for HSA. This fact parallels the slow photomodification of the tryptophyl side chains (see Table 1).
Conformational studies on unirradiated and irradiated samples of BSA and H S A
Exposure of unirradiated BSA to increasing amounts of the denaturant guanidinium chloride caused remarkable perturbations of its fluorescence properties (see Fig. 7). The initial increase of the emission yield should reflect the interaction of guanidine with accessible fluorophores, resulting in a change of the refractive index of the medium (Donovan. 1964); the subsequent depression of the yield is the consequence of the denaturant-induced unfolding of the protein molecule, which brings the originally internal residues into greater contact with the aqueous solvent. The 15 min-irradiated BSA derivative displayed an essentially identical pattern of fluorescence changes upon treatment with guanidinium chloride (Fig. 7); for both proteins, the midpoint of the conformational transition was located at 2.7 M guanidine. On the other hand, the 60 min-irradiated BSA sample was characterized by a much less pronounced fluorescence increase at low denaturant con-
995
Irradiation time (min) Theoryt. 0 15
~
~
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methioninet Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
54 29 23 83 25
a
63 35 39 6 8 61 18 30 59 16 22
60
~~
57.9 28.5 23.1 83.6 25.1 12.4 62.0 24.6 39.6 3.8 7.3 62.9 15.0 29.5 57.8 15.2 21.8
56.9 27.0 24.0 84.1 25.8 11.9 62.6 25.1 39.7 3.9 7.1 60.8 15.2 29.8 59.5 13.5 22.0
57.7 27.6 22.7 83.7 24.8 12.1 63.3 24.6 39.7 3.6 7.8 60.4 12.7 30.8 59.8 10.8 21.1
*The values in the Table are expressed as mol of amino acid per mole of protein. For each sample, phenylalanine. valine and alanine were used as internal standards. ?Taken from Behrens et al. (1975) in the revised version given in the Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, U.S.A., Edition 1976). $The 60min-irradiated sample showed no trace of methionine sulfoxide, i.e. the usual photooxidation product of methionine, when assayed after alkaline hydrolysis (Jori et nl., 1968). centrations; moreover, the midpoint of the guanidineinduced transition occurred at about 1.85 M guanidine. The effect of guanidine concentration on the fluorescence emission intensity of the native and irradiated Table 3. Amino acid composition of bovine serum albumin after different periods of irradiation of its complex with bilirubin* Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine* Isoleucine Leucine Tyrosine Phenylalanine. Lysine Hist idine Arginine
Irradiation time (min) Theoryt 0 15 53 34 28 78 28 15 46 35 36 4 14. 61 19 26 59 17 23
54.7 33.6 30.6 80.3 28.0 16.7 46.8 29.3 35.3 3.3 14.6 63.7 18.0 27.4 60.5 17.0 22.2
55.1 35.0 30.2 79.6 28.3 15.8 46.6 31.1 36.5 3.4 14.9 60.4 17.9 26.8 57.7 15.3 22.6
60 54.2 34.4 30.2 79.8 28.0 15.8 47.2 30.4 34.7 3.0 14.7 62.4 16.1 26.9 60.1 14.7 22.6
*See the footnotes to Table 3. tTaken from Brown (1975) in the revised version given in the Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, U.S.A., Edition 1976).
FIRMINO F. RUBALTELLI and GIULIO JORI
996
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300
L
60 -
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2
3 GUANlOlNE
,. " 1
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A
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-0
o -
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4 CONCENTRATION
5
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6
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Figure 7. The effect of increasing concentrations of guanidinium chloride on the fluorescence emission of unirradiated (0--0). 15 min-irradiated (0-0). and 60 min-irradiated (A A JBSA solutions. The irradiations were carried out as specified for Fig. 6 . Excitation wavelength : 295 nm. lnstrumcntal sensitivity: 10; excitation and emission slits: 6 nm. ~
samples of HSA is shown in Fig. 8. Unirradiated HSA exhibited two denaturant-induced transitions, between 0.5 M and 1.4 M guanidine and above 2.8 M guanidine, respectively. Once again, the 15 min-irradiated derivative yielded a closely similar plot, whereas the two transitions were barely detectable in the case of the 60 min-irradiated derivative. The plots presented in Figs 7 and 8 were obtained with 295 nm-excitation; however, identical plots were obtained also by fluorescence excitation with 278 nm light. which is also significantly absorbed by the tyrosyl residues of the proteins (Burstein et al.. 1973). The changes of BSA and HSA tertiary structure upon bilirubin-sensitized photooxidation were also probed by studying the effect of temperature on the fluorescence emission. In all cases, increasing the temperature provoked a steady decrease of the emission yield, as is usual for most proteins owing to an enhanced non-radiative decay of the excited singlet state (Longworth, 1971). At temperatures above 45'32, a gradual blue shift of the emission maximum took place, indicating a reduced exposure of the tryptophyl
side chains to the aqueous environment. Probably. this fact was a consequence of the aggregation of albumin molecules at relatively high temperatures. since the occurrence of such a process has already been observed for albumin exposed to other denaturing agents (Chen, 1967, 1973). The experimental data can be analyzed (Weinryb and Steiner, 1970) by a semilogarithmic plot reporting the reciprocal of the emission quantum yield Q as a function of T - ' , where T is the absolute temperature; the slope of the linear portion of the plot yields the activation energy for the thermal denaturation of the protein. Our plots for the unirradiated and irradiated derivatives of BSA and HSA are shown in Fig. 9a and b, respectively. The activation energies calculated for 0. 15 and 60min-irradiated BSA samples were 0.75. 0.77 and 1.83 kcal/mol, respectively. For the HSA derivatives, the experimental plots were essentially superimposable with an activation energy of 1.64 kcal/mol. The Q values at the different temperatures were estimated with reference to a quantum yield at 2 0 C of 0.24 for HSA and 0.37 for BSA (Burstein et a/., 1973).
80 I
I
1
I
I
1
2
3
4
5
WANIOINE
CONCENTRATION
(
~
~
6
M)
8. The effect of increasing concentrations of nuanidinium chloride on the fluorescence emission ofimirradiated (0.--.O), 15 mi;-irradiated (e-OL and 60 rnin-irradiated (A-A) HSA solutions. Irradiation and instrumental conditions as specified for Fig. 7.
Serum albumin-bilirubin complex
991
50 70
30 -
10
-
.I
a
t
33
I
3.2
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3.1
3.0
2.9
2.8
3.3
t
X
T
10-3
I
3.2
I
I
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I
3.1
3.0
2.9
28
( )o
Figure 9. The effect of temperature on the fluorescence emission quantum yield of HSA (Fig. 9A) and BSA (Fig. 9B):).---.( unirradiated, (0-0) 15 min-irradiated, and (A-A) 60 minirradiated samples, Instrumental sensitivity: 30; excitation wavelength: 295 nm; excitation slit: 6 nm; emission slit: 8 nm.
DISCUSSION
E@cr of irradiarion on the afinity of bilirubinfor BSA und H S A Albumin-bound bilirubin quenches the fluorescence emission of the protein tryptophyl residues (Chen, 1973. 1974). Assuming that bilirubin is bound only at the high affinity site, one can utilize the fluorescence quenching data to estimate the association constant for the bilirubin-albumin complexes (Chen, 1973). The values so obtained for the various HSA and BSA derivatives are reported in Table 4. Our data for native BSA and HSA are in good agreement with those obtained by other authors, using the fluorescence quenching (Chen, 1973), the peroxidase method (Faerch and Jacobsen, 1975), the circular dichroism measurements (Blauer et al., 1977), under experimental conditions analogous to ours. The irradiated samples were preliminarily freed of bilirubin and its photodegradation products by gel filtration (see Methods); the pooled albumin fractions, whose concentration had been determined by amino acid analysis, were added with suitable amounts of fresh bilirubin and the new quenching efficiency was evaluated by spectrophotofluorimetry.
Photooxidation of proteins in the presence of photosensitizers is well known (Spikes and Livingston, 1969); however, there is some controversy on the capacity of bilirubin to induce the photooxidation of the serum albumin to which it is bound. Our data clearly indicate that both BSA and HSA protect bound bilirubin from photodegradation even in the presence of different photosensitizers. One possible explanation of such photoprotective action might implicate a competition between bilirubin and some amino acid side chains of the protein for the photooxidizing agent(s); as a consequence, the availability of the photoreactive species to attack the bilirubin moiety would be lowered. Although the question was raised by other authors (Ode11 et al., 1970: Behrman et al., 1974; Pedersen et al., 1977), it has been generally assumed that the occurrence of protein photomodification is unlikely, owing to the high reactivity of bilirubin for lo2, coupled with the relatively low efficiency of '02 generation by electronically excited bilirubin (McDonagh, 1971, 1974; Foote and Ching, 1975). However, this assumption is in con-
Table 4. Association constant of bilirubin for the high-affinity binding site on human and bovine serum albumin" Irradiation time (min) Sample HSA BSA
0
2.07 x 10*M-' 2.16 x 1 0 7 ~ - 1
15 0.54 x 10'M-l 0.87 107 M - '
60 0.67 x 108M-1 0.40 x 1 0 7 ~ - '
*The constants were calculated by the fluorescence quenching technique described by Chen (1973).
998
FIRMINO F. RUBALTELLIand GIULIOJORI
trast with the photodamage of various biomolecules induced by bilirubin (Speck, 1974; Girotti, 1975; Engelhardt et al., 1977). Actually, the parameters characterizing ' 0 , generation by and ' 0 , reactivity with bilirubin may no longer obtain for some out of the photodegradation products of the pigment, which often display absorption spectra overlapping with that typical of bilirubin. Thus, the ability of methylvinylmaleimide~ a bilirubin photoproduct, to photogenerate both '02and radical species has been recently documented (Kurtin, 1978). Moreover, react ion mechanisms involving transient species other than '0, (e.g. radical intermediates) have been shown to be involved in bilirubin photochemistry to an extent depending on bilirubin conformation and solvent .polarity (Lightner e? a/., 1976; Manitto e? d., 1972; Manitto and Monti. 1976). Thus, it has been pointed out by Ostrow and Branham (1970) and confirmed by us that albumin-bound bilirubin undergoes photodegradation even in oxygen-free solutions. It is worthwhile noting that the formation of dye-protein ground state complexes prior to irradiation strongly favours the occurrence of type I (radical involving) processes in photosensitized reactions (Bellin and Yankus, 1969: Jori, 1974). It is not surprising, therefore, that we have observed chemical and conformational modifications of both BSA and HSA under visible light-irradiation of their 1: 1 complex with bilirubin. Besides amino acid analyses, other evidence, which unequivocally demonstrates the occurrence of such alterations, is the irradiation time-dependent changes of the light absorption and emission properties of the proteins, the increased sensitivity of the irradiated albumins to denaturation, and their diminished affinity for bilirubin. Similar effects have been observed (Odell et al., 1970) in the case of HSA subjected to methylene bluesensitized photooxidation. O n the other hand, our findings contradict a recent report by Pedersen et al. (1977) stating that HSA was recovered unchanged after 60 min irradiations of its bilirubin complex with eight 20 W/15 Osram fluorescence lamps. However, the total light intensity used by these authors was much weaker than we have used, so that, under their irradiation conditions, remarkably long periods of irradiation are probably necessary to observe photoeffects comparable with ours. Furthermore, Pedersen rt a/. (1977) estimated the histidine content of native and photooxidized albumin on the intact protein by reaction with diazo-1 H-tetrazole. The latter method is appreciably less sensitive for histidines than amino acid analysis and may yield erroneous data owing to the possible parallel attack of the reagent on tyrosyl side chains (Stark, 1970). Our interpretation of the albumin behaviour during the photodegradation of bound bilirubin receives further support from the observation that BSA protects bilirubin also from dye-sensitized photomodification (Fig. 4). All the dyes used are known to photogenerate ' 0 , and/or radical-type intermediates
(Foote, 1976) and can induce the photooxidation of albumin (Spikes and Livingston, 1969). Some of the dyes tested by us, such as hematoporphyrin and riboflavin, are firmly bound by albumin, whereas the cationic dyes methylene blue and acridine orange do not interact with the protein (Kamisaka et al., 1974: Lightner et al., 1976; Pedersen et al., 1977). The initially preferential modification of 1.5-2 histidyl side chains in HSA and BSA is connected with the well-documented spatial selectivity of dye-sensitized photoreactions (Jori. 1974). so that only those amino acid residues which are adjacent to the dye undergo photooxidation in the early stages. This interpretation is in agreement with the conformational investigations carried out on the 15 min-irradiated albumins, showing that the protein derivatives modified at two histidines had a tertiary structure closely similar with that of the unirradiated proteins. Therefore, it appears that, both in HSA and in BSA, two histidyl residues are located at or in close proximity to the bilirubin strong binding site. The importance of two histidyl residues for bilirubin binding by HSA had been previously inferred from the data of chemical (Jacobsen, 1972) and photosensitized (Odell er d., 1970) modification studies. Upon prolonged light exposure, the decrease in the content of other histidyl, as well as of tyrosyl and tryptophyl residues, bzcame significant. It is likely that the integrity of at least some of these amino acid side chains is critical for maintaining the native threedimensional geometry of the albumin molecule; consequently, molecular regions not closely adjacent to the bilirubin binding site became accessible to the photoreactive species. Actually, the 60 min-irradiated BSA and HSA derivatives were largely denatured, as indicated by their enhanced sensitivity to guanidinium chloride and by the increased activation energy for t hermally-induced fluorescence transitions. as is typical of aromatic fluorophores in progressively larger contact with the aqueous solvent (Longworth, 1971; Weinryb and Steiner, 1970). Furthermore, it is possible that some bilirubin photoproducts still possess photosensitizing properties for amino acids, hence they can promote further modifications when bilirubin has been largely degradated; since several bilirubin photoproducts (Ostrow, 1972; Berry e? a/., 1972) are water-soluble, one should expect that they can freely move in the solution, causing a random attack on the potentially photooxidizable amino acid residues. Previous findings also showed that the indole side chains of tryptophans are unimportant for bilirubin binding by albumins (Jacobsen, 1972) and are located at appreciable distances from the high affinity site (Chen, 1974). O n the whole, there are some differences in the number of modified amino acids between HSA and BSA, as well as in the time-course of their spectral changes and. of bound bilirubin photodegradation. This fact probably reflects subtle differences in the topography of the bilirubin binding site on the two
Serum albumin-bilirubin complex proteins, as other authors had already hypothesized on the basis of circular dichroism (Beaven et al., 1973; Harmatz and Blauer, 1975; Blauer et al., 1977), fluorescence emission (Beaven et al., 1973; Chen, 1973; 1974), and light absorption experiments (Chen, 1973; Harmatz and Blauer, 1975; Faerch and Jacobsen, 1977). The same line of evidence is provided by the different stability constants typical of the complexes between bilirubin and unirradiated HSA and BSA (Chen, 1973; Blauer et al., 1977). Therefore, since the observed changes of the native tertiary structure were remarkably greater for photooxidized BSA, the conclusion must be drawn that the overall three-dimensional organization of the BSA molecule is more labile than that of HSA. The interest in the photooxidation of albumin is due to the possible consequent modification and eventual loss of binding capacity. In fact, the cytoxicity of bilirubin seems to be closely related with the concentration of diffusible bilirubin (albumin-free) in extracellular fluids, since albumin serves as the major carrier for bilirubin (Stern, 1974). Consequently, the binding quality of this protein can be critical in determining the eventual presence of diffusible bilirubin. Under our conditions, the association constant of bilirubin for the high affinity site on HSA and BSA was significantly decreased after the specific modification of two histidyl residues. In vivo studies have shown (Cashore et al., 1975% b) that phototherapy does not decrease the total bilirubin binding capacity of serum albumin. However, the light intensities used
999
in our studies are much higher than those used in clinical phototherapy. Thus, further studies are needed to ascertain the real bearing of our data. on phototherapy. I n addition, it appears important to investigate if the three-dimensional organization of fetal HSA is more labile than that of adult HSA, since fetal and adult albumin are thought to have different binding characteristics (Chignell et al., 1971). gONCLUSIONS
1. Some amino acid residues of BSA and HSA are modified by illumination of the albumin-bilirubin complex with high intensity visible light at pH 7.4. Bound bilirubin acts as the photosensitizer in the early stages of the process, causing the selective modification of about two histidyl residues. 2. The two histidyl residues are unimportant for maintaining the native three-dimensional structure of BSA and HSA, but they are critical for determining the binding capacity of the albumins; therefore, they appear to be present at the high affinity binding site of BSA and HSA for bilirubin. 3. The tertiary structure of the BSA molecule is more labile than that of HSA; thus, the effects of bilirubin-sensitized photooxidation are more pronounced for BSA. This observation suggests that the importance of the bilirubin photoeffects depends also on the albumin threedimensional organization. The problem is of interest for comparing the photoeffects of bound bilirubin on adult and fetal HSA.
REFERENCES
Bakken, A. F. (1977) Acta Paediat. 66, 91-95. Beaven, G. H., A. DAlbis and W. B. Gratzer (1973) Eur. J. Biochem. 33, 500-510. Behrens, P. A., A. hp. Spiekerman and J. R. Brown (1975) Fed. Proc. 34, 591. Behrman, R. E., A. K. Brown, M. R. Currie, J. V. Hastings, G. B. Odell, R. Schaffer, R. B. Setlow, T. P. Vogl, R. J. Wurtman. R. J. Anderson, H. J. Kastowski and A. P. Simonpoulos (1974) J. Pediat. 84, 135-147.
Bellin, J. S. and C. A. Yankus (1968) Arch. Biochem. Biophys. 123, 18-31. Berry, C. S., J. E. Zarembo and J. D. Ostrow (1972) Biochem. Biophys. Res. Commun. 49, 1366-1375. Blauer, G., E. Lavie and J. Silfen (1977) Biochim. Biophys. Acta 492, 64-69. Brown, J. R. (1975) Fed. Proc. 34, 591. Burstein, E. A., N. S. Vedenkina and M. N. Ivkova (1973) Photochem, Photobiol. 18, 263-279. Cashore, W. J., E. J. Karotkin and W. Oh (1975a) Pediat. Res. 9, 275. Cashore, W. J., E. J. Karotkin, L. Stern and W. Oh (1975b) J. Pediat. 87, 977-980. Chen. R. F. (1967) J . Biol. Chem. 242, 173-181. Chen. R. F. (1973) In Fluorescence Techniques in Cell Biology (Edited by A. Thaer and M. Sernetz). pp. 239-248. Springer, New York. Chen, R. F. (1974) Arch. Biochem. Biophys. 160, 106-112. Chignell, C. F., E. S. Visell, D. K. Starkweather and C. M. Berlin (1971) Clin. Pharm. Therap. 12, 897-901.
Clark, P., M.R. Rachinsky and J. F. Forster (1962) J. Biol. Chem. 237, 2505L2516. Davies, R. E. and S. J. Keohane (1970) Bull. Chim. Farm. 109, 589-598. Donovan, J. W. (1964) Biochemistry 3, 67-72. Engelhardt, D. L., R. M. Santella, H. S. Rosenkranz and W. T. Speck (1977) Photochem. Photobiol. 26, 53-55.
Faerch, T. and J. Jacobsen (1975) Arch. Biochem. Biophys. 168, 351-357. Faerch, T. and J. Jacobsen (1977) Arch. Biochem. Biophys. 184, 282-289. Foote, C. S. (1976) In Free Radicals in Biology (Edited by W . A. Pryor). Vol. 11, pp. 85-134. Academic Press, New York. Foote. C. S. and T. Y. Ching (1975) J. Am. Chem. Soc. 97. 6209-6214. Genov. N. and G. Jori (1973) I n t . J . Peptide Protein Res. 5, 127-133. Girotti. A. W. (1975) Biochemistry 14. 3377-3383.
I000
FIRMINO F. RURALTELLI and Giu~.ioJOKI Harmatz, D. and G . Blauer (1975) Arch. Biochem. Biophys. 170. 375-383. Jacobsen, J. (1972) Eur. J . Biochem. 27, 513519. Jacobsen, J. (1977) Int. J . Peptide Protein Res. 9, 235-239. Jori, G. (1974) A m i s de Academia Brasileira de Ciencias 45, (suplemento 1973). 33-44. Jori, G., G. Galiauo, A. Marzotto and E. Scoffone (1968) Biochim. Biophys. Actu 154, 1-9. Kamisaka, K., J. Listowsky, J. J. Betheil and J. M. Arias (1974) Biochim. Biophys. Actu 365, 16%180. Kostenbauder, H. B. and D. R. Sanvordeker (1973) Experientia 29, 282-283. Kurtin, W. E. (1978) Photochem. Photobiol. 27, 50S509. Lightner, D. A. (1977) Photochem. Photobiol. 26, 427-436. Lightner, D. A., A. Cu, A. F. McDonagh and L. Palma (1976) Biochem. Biophys. Res. Commurr. 69, 648-657. Longworth, J. (1971) In Excited states of Protein and Nucleic Acid (Edited by R. F. Steiner and J. Weinryb), pp. 319484. Plenum Press, New York. Maehly. A. C. (1961) Nature 192, 630-632. Manitto, P. and D. Monti (1976) J . Chem. Soc. Chem. Commun. 122.123. Manitto, P., D. Monti and E. Garbagnati (1972) I t Farmaco, Ed. Sci. 27, 999-1002. McDonagh. A. F. (I971 ) Biocheni. Biophys. Res. Cornmuti. 44, 1306- I3 1 I . McDonagh. A. F. (1974) In Phototherapy in the Newborn: An Ocerview (Edited by G. B. O d d , R. Schafler and A. P. Simopoulos), pp. 56-73. National Academy of Sciences, Washington, D.C. McDonagh, A. F. and F. Assisi (1972) FEES Lett. 18, 315-317. Moore, S. and W. M. Stein (1963) Methods in Enzynrol. 6, 819-831. Odell, G. B.. R. S. Brown and A. E. Kopelman (1972) J. Pediatr. 81. 473-483. Odell, G. B., R. S. Brown and N. A. Holtzman (1970) In Bilirubin Metubolism in the Newborn (Edited by D. Bergsma. D. Y. Y. Hsia and C. Jackson), pp. 31-36. National Foundation for Birth Defects, New York. Ostrow, J. D. (1972) Sem. Hematol. 9, 113-125. Ostrow J. D. and R. V. Branham (1970) In Bihubin Metabolism in the Newborn (Edited by D. Bergsma. D. Y. Y. Hsia and C. Jackson), pp. 93-99. National Foundation for Birth Defects. New York. Pedersen. A. O., F. Schonbeyder and R. Brodersen (1977) Eur. J . Biochem. 72, 213-221. Rubaltelli, F. F. (1973) Minerua Pediatr. 25, 69-78. Rubaltelli, F. F., G. Allegri, C. Costa and A. De Antoni (1974) J. Pediut. 85, 865-867. Rubaltelli. F. F. and G. Largajolli (1973) Actu Pediut. Scand. 62, 14&148. Sisson, T. R. C. (1976) Photochem. Photobid. Rev. 1, 241-268. Speck, W. T. (1974) Pediat. Res. 8, 451 (abstract). Spikes, J. D. and R. Livingston (1969) Adu. Radiut. Biol. 3, 29-121. Spikes, J. D. and M. L. McKnight (1970) Ann. N.Y. Acad. Sci. 171, 149-162. Stark, G. R. (1970) Adu. Prof. Chem. 24, 261-308. Stern, L. (1974) In Bilirubin Metabolism in the Newborn (11) (Edited by D. Bergsma and S. H. Blondheim), Vol. XII, No. 2, pp. 321-324. National Foundation for Birth Defects, New York. Weinryb, 1. and R. F. Steiner (1970) Biochemistry 9, 135-146.
Photochemistry ond Phorobralogy. Vol. 29. pp. 991 10 IMW). 0 Perg,,rnun Press Lld. 1979. Printcd in Great Brilmn
VISIBLE LIGHT IRRADIATION OF HUMAN AND BOVINE SERUM ALBUMIN-BILIRUBIN COMPLEX* FIRMINO F. RUBALTELLI and G~ULIO JORI Department of Pediatrics and C.N.R. Center for the Physiology and Biochemistry of Hemocyanins and Other Metallo-Proteins, Institute of Animal Biology, University of Padova, Padova, Italy (Received 12 October 1978; accepted 14 December 1978)
Abstract-The UV absorption and the fluorescence emission spectra of both bovine (BSA) and human (HSA) serum albumin underwent noticeable changes upon irradiation of their 1: 1 complexes with bilirubin; both these phenomena are suggestive of the photosensitized modification of aromatic amino acid residues. Amino acid analysis showed that after relatively short irradiation times of both albumins, only histidyl and tryptophyl residues appeared to be affected to a significant extent. After 60min of irradiation, some decrease in the tyrosine content was also observed, especially for HSA. Conformational studies, obtained by exposing unirradiated and irradiated BSA and HSA to denaturing agents, showed that the three-dimensional organization of the 15 min irradiated samples was slightly differentfrom that of the native proteins. On the other hand, after 15 min of irradiation, the association constant of the bilirubin-albumin complexes decreased from 2.07 to 0.54 x IO'M-' for HSA and from 2.16 to 0.87 x LO'M-' for BSA. These data indicate that the histidyl residues are relatively unimportant for maintaining the native tertiary structure of BSA and HSA, but they are critical for determining the binding capacity of the albumins. Our data also imply that the tertiary structure of the BSA molecule is more labile than that of HSA. INTRODUCTION
Phototherapy is frequently used in the treatment of unconjugated hyperbilirubinemia of premature newborns (Rubaltelli, 1973; Sisson, 1976). Present evidence indicates that photodynamic damage can be ruled out as a major hazard of in uiuo light treatment, although some metabolic processes may be altered in jaundiced infants treated by phototherapy (Rubaltelli and Largajolli, 1973; Rubaltelli et al., 1974; Bakken, 1977). However, it is still to be shown that minor photodynamic effects are absent. Since bilirubin is predominantly bound to albumin, it is conceivable that amino acid residues of the albumin molecule are, at least in uitro, the main site for photodynamic attack. Photooxidized albumin exhibits a reduced bilirubin-binding capacity (Cashore et al., 1975a; Odell et al., 1970), which might possibly increase the risk of kernicterus. Cashore et al. (1975b) were able t o demonstrate that phototherapy does not influence the bilirubinalbumin complex in uiuo, at least as studied with Sephadex gel filtration. However, the effect of light treatment on albumin in the presence of bilirubin has not been fully clarified. The pioneering work of Odell et al. (1970) has shown that, in the presence of sensitizing dyes, the imidazole ring of histidine is readily destroyed by light treatment in uitro and, in addition, the bilirubin-albumin binding is decreased, possibly *This work has been supported in part by C.N.R. grants No. 77.01550.64 and No. 78.01876.65 under the C.N.R.N.S.F. cooperative research program.
as a cokequence of alterations of the albumin tertiary structure. O n the contrary, Pedersen et al. (1977) have found that human serum albumin does not undergo in uitro photooxidation upon direct irradiation of its complex with bilirubin and have proposed that one of the possible mechanisms of phototherapy is a Z-E photoisomerization of bilirubin when complexed with albumin to produce a water-soluble compound. Most authors generally agree that the albuminbound bilirubin is photooxidized at a slower rate than unbound bilirubin, although the mechanism of such photoprotection is not fully understood (Lightner, 1977). O n the other hand, Ostrow and Branham (1970) and Ostrow (1972) found that at pH values from 7.4 to 9.9 the albumin addition accelerates the destruction of bilirubin by light, whereas the protein stabilizes the pigment against light at pH 11-13. The present studies were designed to investigate the photodegradation of the bilirubin-albumin complex upon direct irradiation with visible light, as well as upon irradiation in the presence of different photosensitizers. The eventual changes of the bilirubinalbumin complex were also studied. MATERIALS AND METHODS
Materials, Bovine (BSA) and human (HSA) serum albumin, crystallized and lyophilized, Fraction V, were pur-
chased from Sigma; their concentrations were estimated spectrophotometrically assuming extinction values at 279 nm of 6.67 and 5.30 for 1% solutions of BSA and HSA, respectively (Clark et al., 1962). Since defatting the proteins with charcoal in acid solution (Chen, 1967) had no appreci591
992
JORI FIRMINO F. RUBALTELLIand GIULIO
able effect on the photooxidation, the commercial preparations were used as such. Bilirubin (Sigma) was shown by thin layer chromatography (McDonagh and Assisi, 1972) to be essentially constituted by the IXa isomer, and its concentration was determined using a molar absorptivity value of 46.500 at 459nm (Chen, 1973). Methylene blue and riboflavin (Merck). acridine orange and hematoporphyrin (Sigma) were used as received. Guanidine hydrochloride (Merck) was recrystallized once from absolute ethanol and fresh solutions of this compound were prepared daily. Bilirubin and bilirubin-protein complexes were prepared by dissolving the dye in a few drops of 0.1 M NaOH and by adding the desired amount of buffer or of a protein solution in 0.5 M KH2PO4-Na2HPO4 buffer at pH 7.4, ionic strength 0.1. The solutions were maintained in the dark and used within a few h. Final solutions to be irradiated were 0.1 mM in both albumin and bilirubin, and absorption spectral studies showed that bilirubin solutions strictly followed Beer's law, suggesting that the dye was present essentially in a monomeric form; under these conditions, bilirubin is essentially all bound at the single high affinity site of HSA and BSA (Jacobsen, 1977).In all cases, the dark controls were recovered unchanged within the periods of time necessary for the irradiation experiments. Irrcidiulion procedure. Light from a stabilized 1250 W high pressure halogen lamp (Osram) was focused by a parabolic reflector on a 1 cm-thick quartz cuvette containing 3m/ of an air-equilibrated aqueous solution of the sample to be irradiated. A freshly prepared acetone filter was placed in the front of the irradiation cuvette to eliminate all wavelengths below 330nm. The irradiance in the 340-470nm region, as measured with a thermopile. was about 3 W/m2/nm. The temperature of the solutions was maintained at 19 f 1°C by means of thermostated water circulating through the cell holder. In kinetic experiments, aliquots were taken at suitable intervals for spectroscopic and other analysical measurements. When the irradiated solutions contained dyes acting as photosensitizers, at a concentration of 0.1 mg/m/, suitable Bakers interference filters with peak transmission at 404 nm (for riboflavin and hematoporphyrin), 490 nm (for acridine orange) and, 640nm (for methylene blue) were used. Spectroscopic measurements. Absorption spectra were monitored at room temperature by a Perkin-Elmer 402 spectrophotometer. using matched quartz cuvettes with an optical path of either 10 or 1 mm. Absorbance measurements at specific wavelengths were obtained with a Zeiss M4/Q 111 single beam spectrophotometer. Fluorescence emission spectra were recorded by a Perkin-Elmer MPF4 spectrophotofluorimeter, equipped with a 150 W Xe light source and R446 F photomultiplier tube. Quartz cuvettes with an optical path of lOmm were used and the light emission was read at 90" to the incident beam. The temperature of the analyzed solutions could be controlled to + O S T by circulating water; in all cases, the samples were kept at the desired temperature for about 10min prior to measurement, in order to ensure the attainment of thermal equilibrium. The optical density of the various samples was always lower than 0.15 at the excitation wavelength, in order to minimize artifacts due to inner filter or selfabsorption effects. Finally, the spectra were corrected for any fluorescence arising from the solvent system used. On the other hand, no corrections were applied for the wavelengthdependence of the light source emission and detector response. Prior to absorption spectral measurements, the dyes and/or bilirubin were removed by a gel filtration of the unirradiated or irradiated solutions through a column (35 x 1.2 cm) of Sephadex (3-25, using the 0.5 M phosphate buffer as the eluant. Control experiments showed that bilirubin was completely removed from unirradiated albumin; on the other hand, after irradiation of the complex, bilirubin and/or
some photoproducts were not removed from albumin even after denaturation of the protein. Further experiments are now in progress in order to clarify the nature of this apparently photoinduced binding. Amino ucid analyses. The dye- and salt-freed albumin solutions were hydrolyzed by heating at 1 1 0 C for 2 h within evacuated sealed vials in the presence of 6 M HCI. The solvent was then removed by lyophilization and the dried samples were chromatographed on a Carlo Erba 3A27 amino acid analyzer following the method of Moore and Stein (1963). The recovery of the various amino acids was estimated using phenylalanine, valine and alanine as internal standards, since these amino acids are known to be stable to acid hydrolysis and are unaffected by photosensitized oxidation processes (Spikes and MacKnipht. 1970). The content of tryptophan was measured on the intact protein by the spectrophotofluorimetric method described elsewhere (Genov and Jori, 1973). To avoid artifacts arising from the effect of protein conformation on the emission properties of the tryptophyl side chains (Longworth, 1971), the protein samples were denatured by diluting 0.5 m/ of the albumin solution with 2.5 m/ of 7 M guanidinium chloride solution in the same buffer. Control experiments showed that there was a linear relationship between protein concentration and intensity of the 295 nmexcited emission. RESULTS
Photodegradation of bilirubin by direct irradiariori Binding of bilirubin with HSA and BSA was accompanied by the well-known bathochromic shift of the absorption maximum of the pigment and by the appearance of hyperchromicity. Irradiation of either free or albumin-bound bilirubin with polychromatic radiation from the halogen lamp caused a gradual decrease of the absorbsnce in the 450 nm region
10
20
30
IRRAOIATION TIME (MIN )
Figure 1. Time-course of the absorbance decrease at 490 nm observed upon irradiation of free bilirubin (.---a), BSA-bound (0-0) and HSA-bound (A-A) bilirubin.
993
Serum albumin-bilirubin complex
-- I . d
40
2.
t
2 30-
* z W
-
W
y
20-
W
w
I
L
680
640
600
I
I
L
560
520
480
WAVELENGTH
Inm)
Figure 2. Fluorescence emission spectra displayed by 0.1 m M bilirubin solutions in 0.5 M phosphate buffer. pH 7.4, which had been irradiated for different periods of time (min). Excitation wavelength: 480 nm. Instrumental Sensitivity: 30; excitation slit: 8 nm: emission slit: 10 nm. and a concomitant enhancement of the absorbance below 350nm, in agreement with the observations of several other authors (see, for a review, Lightner, 1977). The time-course of light induced decrease of bilirubin absorption at 490nm is shown in Fig. 1; this wavelength was chosen since previous studies pointed out that at 490 nm little interference occurs from the bilirubin photodegradation products (Davies and Keohane, 1970; Ostrow, 1972). Essentially identical plots were obtained if the absorbance changes were followed at 460 nm. Clearly, the spectral changes were less pronounced for any given period of irradiation when bilirubin was complexed with HSA or BSA. Further information on bilirubin degradation was obtained by fluorescence emission studies. As shown by other authors (Beaven et a!., 1973; Chen, 1973), binding of bilirubin to albumins results in a remarkI
I
c
A
B
I
20 IRRAOIATION TIME (min 1 10
30 TIME
Figure 3. Time-course of the emission intensity enhancement at 540 nm observed upon irradiation of free bilirubin ( 0 . -0).
able enhancement of the intrinsically low fluorescence of the pigment around 530 nm. We have reconfirmed these data. Moreover, we observed that, upon irradiation, the yield of the 480 nm-excited fluorescence emission increases (see, for example, Fig. 2), although the rate and the extent of the fluorescence enhancement were again different for free and protein-bound bilirubin (Fig. 3); in particular, for HSA-bound bilirubin, no appreciable change of the fluorescence yield was observed after about 10min of irradiation. As pointed out above, the fluorescence quantum yield of free bilirubin is very low; therefore, the emission enhancement should not reflect a dissociation on the bilirubin-albumin complex. Rather, it might be connected with the formation of bilirubin photoisomers
BSA-bound
(0-O),
and
HSA-bound
(A --A)bilirubin under the experimental and instrumental conditions specified for Fig. 2.
(MINI
Figure 4. Time-course of 0.1 mM bilirubin photodegradahematoporphyrin tion sensitized by riboflavin (0-0). (A-A), methylene blue (a-W), and acridine orange (o-o), as deduced from the absorbance decrease at 490 nm. A: free bilirubin, B: BSA-bound bilirubin.
994
FIRMINO
F. RUBALTELLIand GIIJLIO JORI
(McDonagh, 1971; Ostrow, 1972; Kostenbauder and Sanvordeker, 1973; Lightner, 1977). We have also studied the disappearance of free and albumin-bound bilirubin, photosensitized by a variety of dyes, under our experimental conditions (See Fig. 4a and b). Since such irradiation was carried out with narrow light bands and under conditions ensuring total absorption of the incident light by the added dye, one can conclude that BSA also protects bound bilirubin from photosensitized degradation. O n the other hand, it is not possible to compare the kinetic plots obtained with the different photosensitizers, since no correction was applied for the quantum efficiency of the light emission by the halogen lamp as a function of the wavelength.
1001
Bilirubin-sensitized photooxidation of albumins
250
290
270
310
WAVELENGTH (nm)
Figure 5. Ultraviolet absorption spectra of 0.05 mM BSA solutions after 0 (.---a), 15 (0-O), and 60 (A-A) minutes of irradiation of their 1:1 complex with bilirubin. Bilirubin and its photoproducts had been previously removed by gel filtration. (Lightner, 1977; Pedersen et a/., 1977) and/or with the formation of fluorescent bilirubin photodegradation products, which have been actually detected in developed chromatograms of irradiated bilirubin solutions (Ostrow, 1972). Photosensitixd deyrudution of bilirubin
Bilirubin degradation can be photosensitized by the addition of suitable dyes to the irradiated system
The UV absorption spectrum of both BSA and HSA underwent noticeable changes upon prolonged irradiation of their 1:l complexes with bilirubin as shown in Fig. 5: the alterations are suggestive of the photosensitized modification of aromatic amino acid residues (Spikes and Livingston, 1969). The same line of evidence was provided by studies of the intrinsic fluorescence emission of the albumins as a function of the irradiation time: as one can see from Figs. 6a and b, prolonging the light-exposure caused a steady decrease of the emission yield of both BSA and HSA. Since the exciting radiation (295 nm) was selectively absorbed by the tryptophyl chromophores. this phenomenon must be the consequence of a gradual photooxidative modification of the indole side chains and/or of conformational transitions of the albumin molecule leading to perturbations of the microenvironment of the tryptophyl residues. In actual fact, quantitative determination of Trp by spectrophotofluorimetric analysis on the unirradiated and irradiated albumin samples. after denatu-
A
420
380
420
340 WAVELENGTH
380
340
trim)
Figure 6. Fluorescence emission spectra of 0.1 mM HSA (a) and BSA (b) solutions in 0.5 M phosphate buffer, pH 7.4, after irradiation of their 1 : 1 complex with bilirubin. Excitation wavelength: 295 nm. Instrumental sensitivity: I ; excitation and emission slits: 6 nm.
Serum albumin-bilirubin complex Table 1. Tryptophan content of human and bovine serum albumin after different periods of irradiation of their complex with bilirubin* Irradiation time (min) ~~
~
Theoryt
-
0 15 60
Table 2. Amino acid composition of human serum albumin after different periods of irradiation of its complex with bilirubin* ~
~~
Amino acid HSA
BSA
1.o 1 .o 0.8 0.5
2.0 1.9 1.4 0.8
~~
*Determined according to the spectrophotofluorimetric procedure described by Genov and Jori (1973). The values in the table are expressed as mol of amino acid per mole of protein. tTaken from Behrens et 01. (1975) for HSA and from Brown (1975) for BSA. ration by 7 M guanidinium chloride, gave the results summarized in Table 1. O n the basis of the above reported studies, protein samples were isolated after 15 and 60min of irradiation for complete amino acid analysis. The data obtained for the unirradiated samples were compared with available reports of the amino acid sequence and composition of HSA (Behrens et al., 1975) and BSA (Brown, 1975), as revised in the 1976 edition of the Atlas of Protein Sequence and Structure published by the National Biomedical Research Foundation, U.S.A. As one can see from Tables 2 and 3, after relatively short irradiation times of both albumins, only histidyl residues appeared to be affected to a significant extent. After 60 min of irradiation, some decrease in the tyrosine content was also observed, especially for HSA. This fact parallels the slow photomodification of the tryptophyl side chains (see Table 1).
Conformational studies on unirradiated and irradiated samples of BSA and H S A
Exposure of unirradiated BSA to increasing amounts of the denaturant guanidinium chloride caused remarkable perturbations of its fluorescence properties (see Fig. 7). The initial increase of the emission yield should reflect the interaction of guanidine with accessible fluorophores, resulting in a change of the refractive index of the medium (Donovan. 1964); the subsequent depression of the yield is the consequence of the denaturant-induced unfolding of the protein molecule, which brings the originally internal residues into greater contact with the aqueous solvent. The 15 min-irradiated BSA derivative displayed an essentially identical pattern of fluorescence changes upon treatment with guanidinium chloride (Fig. 7); for both proteins, the midpoint of the conformational transition was located at 2.7 M guanidine. On the other hand, the 60 min-irradiated BSA sample was characterized by a much less pronounced fluorescence increase at low denaturant con-
995
Irradiation time (min) Theoryt. 0 15
~
~
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methioninet Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
54 29 23 83 25
a
63 35 39 6 8 61 18 30 59 16 22
60
~~
57.9 28.5 23.1 83.6 25.1 12.4 62.0 24.6 39.6 3.8 7.3 62.9 15.0 29.5 57.8 15.2 21.8
56.9 27.0 24.0 84.1 25.8 11.9 62.6 25.1 39.7 3.9 7.1 60.8 15.2 29.8 59.5 13.5 22.0
57.7 27.6 22.7 83.7 24.8 12.1 63.3 24.6 39.7 3.6 7.8 60.4 12.7 30.8 59.8 10.8 21.1
*The values in the Table are expressed as mol of amino acid per mole of protein. For each sample, phenylalanine. valine and alanine were used as internal standards. ?Taken from Behrens et al. (1975) in the revised version given in the Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, U.S.A., Edition 1976). $The 60min-irradiated sample showed no trace of methionine sulfoxide, i.e. the usual photooxidation product of methionine, when assayed after alkaline hydrolysis (Jori et nl., 1968). centrations; moreover, the midpoint of the guanidineinduced transition occurred at about 1.85 M guanidine. The effect of guanidine concentration on the fluorescence emission intensity of the native and irradiated Table 3. Amino acid composition of bovine serum albumin after different periods of irradiation of its complex with bilirubin* Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine* Isoleucine Leucine Tyrosine Phenylalanine. Lysine Hist idine Arginine
Irradiation time (min) Theoryt 0 15 53 34 28 78 28 15 46 35 36 4 14. 61 19 26 59 17 23
54.7 33.6 30.6 80.3 28.0 16.7 46.8 29.3 35.3 3.3 14.6 63.7 18.0 27.4 60.5 17.0 22.2
55.1 35.0 30.2 79.6 28.3 15.8 46.6 31.1 36.5 3.4 14.9 60.4 17.9 26.8 57.7 15.3 22.6
60 54.2 34.4 30.2 79.8 28.0 15.8 47.2 30.4 34.7 3.0 14.7 62.4 16.1 26.9 60.1 14.7 22.6
*See the footnotes to Table 3. tTaken from Brown (1975) in the revised version given in the Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, U.S.A., Edition 1976).
FIRMINO F. RUBALTELLI and GIULIO JORI
996
-
300
L
60 -
I'
I
I
I
1
2
3 GUANlOlNE
,. " 1
n a
A
- -
-0
o -
0
A
I
1
4 CONCENTRATION
5
I
6
(M 1
Figure 7. The effect of increasing concentrations of guanidinium chloride on the fluorescence emission of unirradiated (0--0). 15 min-irradiated (0-0). and 60 min-irradiated (A A JBSA solutions. The irradiations were carried out as specified for Fig. 6 . Excitation wavelength : 295 nm. lnstrumcntal sensitivity: 10; excitation and emission slits: 6 nm. ~
samples of HSA is shown in Fig. 8. Unirradiated HSA exhibited two denaturant-induced transitions, between 0.5 M and 1.4 M guanidine and above 2.8 M guanidine, respectively. Once again, the 15 min-irradiated derivative yielded a closely similar plot, whereas the two transitions were barely detectable in the case of the 60 min-irradiated derivative. The plots presented in Figs 7 and 8 were obtained with 295 nm-excitation; however, identical plots were obtained also by fluorescence excitation with 278 nm light. which is also significantly absorbed by the tyrosyl residues of the proteins (Burstein et al.. 1973). The changes of BSA and HSA tertiary structure upon bilirubin-sensitized photooxidation were also probed by studying the effect of temperature on the fluorescence emission. In all cases, increasing the temperature provoked a steady decrease of the emission yield, as is usual for most proteins owing to an enhanced non-radiative decay of the excited singlet state (Longworth, 1971). At temperatures above 45'32, a gradual blue shift of the emission maximum took place, indicating a reduced exposure of the tryptophyl
side chains to the aqueous environment. Probably. this fact was a consequence of the aggregation of albumin molecules at relatively high temperatures. since the occurrence of such a process has already been observed for albumin exposed to other denaturing agents (Chen, 1967, 1973). The experimental data can be analyzed (Weinryb and Steiner, 1970) by a semilogarithmic plot reporting the reciprocal of the emission quantum yield Q as a function of T - ' , where T is the absolute temperature; the slope of the linear portion of the plot yields the activation energy for the thermal denaturation of the protein. Our plots for the unirradiated and irradiated derivatives of BSA and HSA are shown in Fig. 9a and b, respectively. The activation energies calculated for 0. 15 and 60min-irradiated BSA samples were 0.75. 0.77 and 1.83 kcal/mol, respectively. For the HSA derivatives, the experimental plots were essentially superimposable with an activation energy of 1.64 kcal/mol. The Q values at the different temperatures were estimated with reference to a quantum yield at 2 0 C of 0.24 for HSA and 0.37 for BSA (Burstein et a/., 1973).
80 I
I
1
I
I
1
2
3
4
5
WANIOINE
CONCENTRATION
(
~
~
6
M)
8. The effect of increasing concentrations of nuanidinium chloride on the fluorescence emission ofimirradiated (0.--.O), 15 mi;-irradiated (e-OL and 60 rnin-irradiated (A-A) HSA solutions. Irradiation and instrumental conditions as specified for Fig. 7.
Serum albumin-bilirubin complex
991
50 70
30 -
10
-
.I
a
t
33
I
3.2
I
I
I
I
I
3.1
3.0
2.9
2.8
3.3
t
X
T
10-3
I
3.2
I
I
I
I
3.1
3.0
2.9
28
( )o
Figure 9. The effect of temperature on the fluorescence emission quantum yield of HSA (Fig. 9A) and BSA (Fig. 9B):).---.( unirradiated, (0-0) 15 min-irradiated, and (A-A) 60 minirradiated samples, Instrumental sensitivity: 30; excitation wavelength: 295 nm; excitation slit: 6 nm; emission slit: 8 nm.
DISCUSSION
E@cr of irradiarion on the afinity of bilirubinfor BSA und H S A Albumin-bound bilirubin quenches the fluorescence emission of the protein tryptophyl residues (Chen, 1973. 1974). Assuming that bilirubin is bound only at the high affinity site, one can utilize the fluorescence quenching data to estimate the association constant for the bilirubin-albumin complexes (Chen, 1973). The values so obtained for the various HSA and BSA derivatives are reported in Table 4. Our data for native BSA and HSA are in good agreement with those obtained by other authors, using the fluorescence quenching (Chen, 1973), the peroxidase method (Faerch and Jacobsen, 1975), the circular dichroism measurements (Blauer et al., 1977), under experimental conditions analogous to ours. The irradiated samples were preliminarily freed of bilirubin and its photodegradation products by gel filtration (see Methods); the pooled albumin fractions, whose concentration had been determined by amino acid analysis, were added with suitable amounts of fresh bilirubin and the new quenching efficiency was evaluated by spectrophotofluorimetry.
Photooxidation of proteins in the presence of photosensitizers is well known (Spikes and Livingston, 1969); however, there is some controversy on the capacity of bilirubin to induce the photooxidation of the serum albumin to which it is bound. Our data clearly indicate that both BSA and HSA protect bound bilirubin from photodegradation even in the presence of different photosensitizers. One possible explanation of such photoprotective action might implicate a competition between bilirubin and some amino acid side chains of the protein for the photooxidizing agent(s); as a consequence, the availability of the photoreactive species to attack the bilirubin moiety would be lowered. Although the question was raised by other authors (Ode11 et al., 1970: Behrman et al., 1974; Pedersen et al., 1977), it has been generally assumed that the occurrence of protein photomodification is unlikely, owing to the high reactivity of bilirubin for lo2, coupled with the relatively low efficiency of '02 generation by electronically excited bilirubin (McDonagh, 1971, 1974; Foote and Ching, 1975). However, this assumption is in con-
Table 4. Association constant of bilirubin for the high-affinity binding site on human and bovine serum albumin" Irradiation time (min) Sample HSA BSA
0
2.07 x 10*M-' 2.16 x 1 0 7 ~ - 1
15 0.54 x 10'M-l 0.87 107 M - '
60 0.67 x 108M-1 0.40 x 1 0 7 ~ - '
*The constants were calculated by the fluorescence quenching technique described by Chen (1973).
998
FIRMINO F. RUBALTELLIand GIULIOJORI
trast with the photodamage of various biomolecules induced by bilirubin (Speck, 1974; Girotti, 1975; Engelhardt et al., 1977). Actually, the parameters characterizing ' 0 , generation by and ' 0 , reactivity with bilirubin may no longer obtain for some out of the photodegradation products of the pigment, which often display absorption spectra overlapping with that typical of bilirubin. Thus, the ability of methylvinylmaleimide~ a bilirubin photoproduct, to photogenerate both '02and radical species has been recently documented (Kurtin, 1978). Moreover, react ion mechanisms involving transient species other than '0, (e.g. radical intermediates) have been shown to be involved in bilirubin photochemistry to an extent depending on bilirubin conformation and solvent .polarity (Lightner e? a/., 1976; Manitto e? d., 1972; Manitto and Monti. 1976). Thus, it has been pointed out by Ostrow and Branham (1970) and confirmed by us that albumin-bound bilirubin undergoes photodegradation even in oxygen-free solutions. It is worthwhile noting that the formation of dye-protein ground state complexes prior to irradiation strongly favours the occurrence of type I (radical involving) processes in photosensitized reactions (Bellin and Yankus, 1969: Jori, 1974). It is not surprising, therefore, that we have observed chemical and conformational modifications of both BSA and HSA under visible light-irradiation of their 1: 1 complex with bilirubin. Besides amino acid analyses, other evidence, which unequivocally demonstrates the occurrence of such alterations, is the irradiation time-dependent changes of the light absorption and emission properties of the proteins, the increased sensitivity of the irradiated albumins to denaturation, and their diminished affinity for bilirubin. Similar effects have been observed (Odell et al., 1970) in the case of HSA subjected to methylene bluesensitized photooxidation. O n the other hand, our findings contradict a recent report by Pedersen et al. (1977) stating that HSA was recovered unchanged after 60 min irradiations of its bilirubin complex with eight 20 W/15 Osram fluorescence lamps. However, the total light intensity used by these authors was much weaker than we have used, so that, under their irradiation conditions, remarkably long periods of irradiation are probably necessary to observe photoeffects comparable with ours. Furthermore, Pedersen rt a/. (1977) estimated the histidine content of native and photooxidized albumin on the intact protein by reaction with diazo-1 H-tetrazole. The latter method is appreciably less sensitive for histidines than amino acid analysis and may yield erroneous data owing to the possible parallel attack of the reagent on tyrosyl side chains (Stark, 1970). Our interpretation of the albumin behaviour during the photodegradation of bound bilirubin receives further support from the observation that BSA protects bilirubin also from dye-sensitized photomodification (Fig. 4). All the dyes used are known to photogenerate ' 0 , and/or radical-type intermediates
(Foote, 1976) and can induce the photooxidation of albumin (Spikes and Livingston, 1969). Some of the dyes tested by us, such as hematoporphyrin and riboflavin, are firmly bound by albumin, whereas the cationic dyes methylene blue and acridine orange do not interact with the protein (Kamisaka et al., 1974: Lightner et al., 1976; Pedersen et al., 1977). The initially preferential modification of 1.5-2 histidyl side chains in HSA and BSA is connected with the well-documented spatial selectivity of dye-sensitized photoreactions (Jori. 1974). so that only those amino acid residues which are adjacent to the dye undergo photooxidation in the early stages. This interpretation is in agreement with the conformational investigations carried out on the 15 min-irradiated albumins, showing that the protein derivatives modified at two histidines had a tertiary structure closely similar with that of the unirradiated proteins. Therefore, it appears that, both in HSA and in BSA, two histidyl residues are located at or in close proximity to the bilirubin strong binding site. The importance of two histidyl residues for bilirubin binding by HSA had been previously inferred from the data of chemical (Jacobsen, 1972) and photosensitized (Odell er d., 1970) modification studies. Upon prolonged light exposure, the decrease in the content of other histidyl, as well as of tyrosyl and tryptophyl residues, bzcame significant. It is likely that the integrity of at least some of these amino acid side chains is critical for maintaining the native threedimensional geometry of the albumin molecule; consequently, molecular regions not closely adjacent to the bilirubin binding site became accessible to the photoreactive species. Actually, the 60 min-irradiated BSA and HSA derivatives were largely denatured, as indicated by their enhanced sensitivity to guanidinium chloride and by the increased activation energy for t hermally-induced fluorescence transitions. as is typical of aromatic fluorophores in progressively larger contact with the aqueous solvent (Longworth, 1971; Weinryb and Steiner, 1970). Furthermore, it is possible that some bilirubin photoproducts still possess photosensitizing properties for amino acids, hence they can promote further modifications when bilirubin has been largely degradated; since several bilirubin photoproducts (Ostrow, 1972; Berry e? a/., 1972) are water-soluble, one should expect that they can freely move in the solution, causing a random attack on the potentially photooxidizable amino acid residues. Previous findings also showed that the indole side chains of tryptophans are unimportant for bilirubin binding by albumins (Jacobsen, 1972) and are located at appreciable distances from the high affinity site (Chen, 1974). O n the whole, there are some differences in the number of modified amino acids between HSA and BSA, as well as in the time-course of their spectral changes and. of bound bilirubin photodegradation. This fact probably reflects subtle differences in the topography of the bilirubin binding site on the two
Serum albumin-bilirubin complex proteins, as other authors had already hypothesized on the basis of circular dichroism (Beaven et al., 1973; Harmatz and Blauer, 1975; Blauer et al., 1977), fluorescence emission (Beaven et al., 1973; Chen, 1973; 1974), and light absorption experiments (Chen, 1973; Harmatz and Blauer, 1975; Faerch and Jacobsen, 1977). The same line of evidence is provided by the different stability constants typical of the complexes between bilirubin and unirradiated HSA and BSA (Chen, 1973; Blauer et al., 1977). Therefore, since the observed changes of the native tertiary structure were remarkably greater for photooxidized BSA, the conclusion must be drawn that the overall three-dimensional organization of the BSA molecule is more labile than that of HSA. The interest in the photooxidation of albumin is due to the possible consequent modification and eventual loss of binding capacity. In fact, the cytoxicity of bilirubin seems to be closely related with the concentration of diffusible bilirubin (albumin-free) in extracellular fluids, since albumin serves as the major carrier for bilirubin (Stern, 1974). Consequently, the binding quality of this protein can be critical in determining the eventual presence of diffusible bilirubin. Under our conditions, the association constant of bilirubin for the high affinity site on HSA and BSA was significantly decreased after the specific modification of two histidyl residues. In vivo studies have shown (Cashore et al., 1975% b) that phototherapy does not decrease the total bilirubin binding capacity of serum albumin. However, the light intensities used
999
in our studies are much higher than those used in clinical phototherapy. Thus, further studies are needed to ascertain the real bearing of our data. on phototherapy. I n addition, it appears important to investigate if the three-dimensional organization of fetal HSA is more labile than that of adult HSA, since fetal and adult albumin are thought to have different binding characteristics (Chignell et al., 1971). gONCLUSIONS
1. Some amino acid residues of BSA and HSA are modified by illumination of the albumin-bilirubin complex with high intensity visible light at pH 7.4. Bound bilirubin acts as the photosensitizer in the early stages of the process, causing the selective modification of about two histidyl residues. 2. The two histidyl residues are unimportant for maintaining the native three-dimensional structure of BSA and HSA, but they are critical for determining the binding capacity of the albumins; therefore, they appear to be present at the high affinity binding site of BSA and HSA for bilirubin. 3. The tertiary structure of the BSA molecule is more labile than that of HSA; thus, the effects of bilirubin-sensitized photooxidation are more pronounced for BSA. This observation suggests that the importance of the bilirubin photoeffects depends also on the albumin threedimensional organization. The problem is of interest for comparing the photoeffects of bound bilirubin on adult and fetal HSA.
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